(1) The arterio-venous difference technique, previously used to measure mammary substrate uptake with cows and goats, was used to measure amino acid uptake by the mammary gland of the lactating Merino ewe. Possession of a single large superficial epigastric vein by the Merino ewe makes the Merino breed the most suitable for this type of study. (2) A method was developed enabling hourly measurement of milk yield without causing undue stress. Milk yield was essentially constant over a 7-8 h period. (3) Mammary extraction of most non-essential amino acids was low relative to output in milk protein and showed a greater variability with time than that found for the essential amino acids. There was a significant mammary extraction of ornithine and citrulline, neither amino acid being found in ovine milk protein. (4) Of the essential amino acids valine, isoleucine, leucine and arginine were taken up in excess of their requirement for milk protein synthesis. (5) On the basis of the extent of mammary extraction, methionine, lysine and leucine were first-, second- and third-limiting to the rate of milk protein synthesis. (6) Despite fluctuations in arterial amino acid concentrations the arterio-venous differences of the essential amino acids were relatively constant over a 7-8 h period. (7) The pattern of mammary amino acid uptake in the ewe is contrasted with that found in similar studies carried out with the lactating cow and goat.